Project Summary Overexpression of multidrug transporters leading to increased, broad-spectrum drug resistance is a major medical problem in the treatment of pathogens and malignant tumors. We use biochemical and genetic approaches to study Pdr5, a major yeast efflux pump that is the founding member of a large subfamily of clinically important fungal ABC transporters. This proposal focuses on a central issue common to all ABC exporters. Once a toxic drug is expelled from the cell, how is it prevented from reentry via the drug-binding sites which are now facing in an extracellular direction? A simple 10-30 fold reduction in affinity which is typically seen during the transport cycle is not sufficient to preclude reflux. Work in our laboratory established that Pdr5 transport is unidirectional and that this efflux pump is therefore a molecular diode. We also identified one residue, Ser-1368 that is essential for diode function. A substitution creating a S1368A mutant shows considerable reflux when a novel diode assay is performed. The present proposal has two aims: First, using a combination of mutant suppression and site-directed mutagenesis, we will identify additional residues that make up the Pdr5 diode. These mutants will be carefully characterized using several biochemical assays including one that is specific for diode function. A central issue is whether both halves of this transporter participate in diode formation or whether like several other biochemical processes diode function is asymmetric. The second aim of the proposal is to biochemically characterize the diode. For instance, we will determine whether there is any substrate specificity to diode operation. The role of ATP hydrolysis and the well-established Pdr5 transmission interface in diode function will be evaluated.